We propose to acquire a FEI Krios 300 kV transmission electron cryo-microscope equipped with a counting mode electron detector, a phase plate holder, a Gatan post-column energy filter with a K2-XP camera and automated data acquisition software. This facility will be administered by the National Center for Macromolecular Imaging (NCMI), a NIGMS supported Biomedical Technology Resource Center at Baylor College of Medicine. In addition to technology development, one of the NCMI's primary missions is to collaborate with and provide service to researchers who need Cryo-electron microscopy (CryoEM) and Cryo-electron tomography (CryoET) for structure determinations from macromolecules to cells at atomic to nanometer resolutions. We have identified 7 major users and 10 minor users in this proposal who are already active NCMI users both in and outside the Houston Area. This instrument facility is aimed for our users who have projects with one or more of the following objectives: (1) atomic resolution structure determination of molecular machines, (2) single particles structures with conformational and/or compositional heterogeneity, (3) subcellular and macromolecular studies of cells in normal and pathological states. All of these users' projects require high throughput and high quality data which will lead to 3-D structures yielding new structural insights of molecular and cellular components and biological processes related to cancer, infectious and neurological diseases. Due to the large quantity of data needed for each of these projects, we will manage our facility similar to an X-ray synchrotron beamline to provide 24 hours per day and 6 days per week (24/6) operation. None of the existing NCMI electron microscopes can deliver comparable data quality with the high throughput needed by the current state of art of CryoEM/ET projects. The actual microscope scheduling will be made quarterly by considering users' preference and the microscope availability. The proposed instrument will be maintained and operated by two NCMI staff who will assist users with data collection. We will provide regular training courses to users and their lab members to enable them to collect data with minimal assistance from our staff. We plan to make the proposed instrument available to other NCMI users after the first 18 months of operation. Then, an external user project evaluation committee made of CryoEM/ET experts appointed by the PI will evaluate and rank the proposals submitted by existing and new users. Two advisory committees will be set up to monitor the operation and productivity of the proposed instrument. One local committee will be appointed by the Senior Vice President of Research at Baylor College of Medicine; while an external committee is set up by the PI as mandated by the NIGMS supported P41 Center. The total cost of the instrument is ~$6 million. Baylor College of Medicine is committed to provide matching funds of $4 million towards the purchase of the instrument and the cost for the laboratory renovation. We have the adequate NIH support for the operation of this proposed instrument via the existing P41 Center grant funding the NCMI, now in its 30th year, extending at least until January 1, 2020. We expect to seek competitive renewal of this grant to continue the operation of the proposed facility beyond 2020. Should this prove unsuccessful, we plan for the proposed instrument to become a Core Lab under the management of the Advanced Technology Core Laboratories (ATCL) at Baylor College of Medicine, which will provide half of the instrument operational costs (service contract, personnel and supplies) and use established ATCL cost recharge mechanisms to recover the remaining operational costs.